The objective of this project is a detailed understanding of formation and maintenance of a dormant, resistant bacterial (Bacillus subtilis) spore; there are 3 major goals. I. The structure of the complex between alpha/beta type small, acid-soluble spore proteins (SASP) and DNA will be determined. Specific aims are: a) probe DNA structure in a SASP/DNA complex; b) identify residues in a SASP/DNA complex involved in SASP-DNA or SASP-SASP interaction; c) study SASP's ability to associate two DNA helices side-by- side; d) use a SASP variant containing trp to study SASP- DNA interaction; e) construct a minimal functional SASP; f) continue collaborative efforts to obtain a high-resolution structure of a SASP/oligonucleotide complex; g) confirm this structure by analysis of appropriate mutant SASP; h) generate SASP altered in the site recognized and cleaved by the SASP specific protease (GPR), analyze their function in vivo, and their cleavage by GPR; and i) identify and characterize minor B. subtilis SASP and the genes coding for them. Il. The fall in intraforespore pH late in sporulation will be studied in detail. Specific aims are: a) analyze the pH decrease in both wild type and spo mutants of B. subtilis; b) modulate depots of 3-phosphoglycerate (3PGA) in vegetative and sporulating cells and germinating spores by altering intracellular pH; c) analyze the effects of inactivation of the B. subtilis pgm gene (coding for phosphoglycerate mutase (PGM)) on growth, sporulation and spore 3PGA depots; d) overexpress B. subtilis PGM or a Mn independent PGM in B. subtilis and analyze the effect of overexpression on spore 3PGA depots; and e) determine the reason for the Mn dependence and pH sensitivity of B. subtilis PGM. III. Analysis of genes and proteins involved in peptidoglycan metabolism during sporulation and germination will be continued. Specific aims are: a) analyze the sequence, regulation and function of additional genes encoding penicillin-binding proteins (PBP); b) with strains lacking one or more PBPs, analyze the cortex structure and heat resistance of their spores; and c) identify the enzyme(s) initiating cortex hydrolysis during spore germination. While this work will be carried out with B. subtilis, the knowledge gained will be applicable to other Gram-positive spore formers. A number of the latter organisms are significant agents of food spoilage as well as human disease, in particular food born disease, in large part because of the resistant nature of spores. Consequently, knowledge gained in the proposed work could have significant applications.